Lithogenic Diet Research Articles

Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice

G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1(-/-) mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1(-/-) mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1(-/-) mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1(-/-) mice on HFD displayed increased hepatic steatosis when compared with Gpbar1(+)(/)(+) males and Gpbar1(-/-) females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.

Gallstones play a significant role inSalmonellaspp. gallbladder colonization and carriage

Salmonella enterica serovar Typhi can colonize the gallbladder and persist in an asymptomatic carrier state that is frequently associated with the presence of gallstones. We have shown that salmonellae form bile-mediated biofilms on human gallstones and cholesterol-coated surfaces in vitro. Here, we test the hypothesis that biofilms on cholesterol gallbladder stones facilitate typhoid carriage in mice and men. Naturally resistant (Nramp1(+/+)) mice fed a lithogenic diet developed cholesterol gallstones that supported biofilm formation during persistent serovar Typhimurium infection and, as a result, demonstrated enhanced fecal shedding and enhanced colonization of gallbladder tissue and bile. In typhoid endemic Mexico City, 5% of enrolled cholelithiasis patients carried serovar Typhi, and bacterial biofilms could be visualized on gallstones from these carriers whereas significant biofilms were not detected on gallstones from Escherichia coli infected gallbladders. These findings offer direct evidence that gallstone biofilms occur in humans and mice, which facilitate gallbladder colonization and shedding.

Comparison of Cholesterol Lowering Diets: Apple, Casein Cytochrom P450 protein and Cholesterol 7α Hydroxylase Activities in Hamsters

Lithogenic diet, casein and apple fiber diets were fed to hamsters for 3-5 weeks. For control group, animals were fed on normal Purina chow without any supplement. The cholesterol lowering effect of lithogenic diet, casein and apple diets were compared. After dietary regimen, animals were screened for any gall stone formation. The isolated liver microsomes were separated from animals and tested for the cholesterol-7α Hydroxylase (CH) enzyme activity measurement in all three groups. The control animals did not show any gall stone formation and their CH enzyme activities were normal. The lithogenic diet showed significantly enhanced CH enzyme activities while animals fed on casein and apple diet regimen showed moderate increase in microsomal CH enzyme activity indicated cholesterol lowering in liver. In conclusion, cholesterol 7α hydroxylase may be a biomarker of cholesterol status in the body and microsomal CH enzyme may be lowered down after treatment of casein and apple diets.

Comparison of Cholesterol Lowering Diets: Apple, Casein Cytochrom P450 protein and Cholesterol 7α Hydroxylase Activities in Hamsters

AbstractLithogenic diet, casein and apple fiber diets were fed to hamsters for 3-5 weeks. For control group, animals were fed on normal Purina chow without any supplement. The cholesterol lowering effect of lithogenic diet, casein and apple diets were compared. After dietary regimen, animals were screened for any gall stone formation. The isolated liver microsomes were separated from animals and tested for the cholesterol-7α Hydroxylase (CH) enzyme activity measurement in all three groups. The control animals did not show any gall stone formation and their CH enzyme activities were normal. The lithogenic diet showed significantly enhanced CH enzyme activities while animals fed on casein and apple diet regimen showed moderate increase in microsomal CH enzyme activity indicated cholesterol lowering in liver. In conclusion, cholesterol 7α hydroxylase may be a biomarker of cholesterol status in the body and microsomal CH enzyme may be lowered down after treatment of casein and apple diets.

Regression of preestablished cholesterol gallstones by dietary garlic and onion in experimental mice

We have recently reported the health beneficial potential of dietary garlic and onion in reducing the incidence and severity of cholesterol gallstone (CGS) during its experimental induction in mice. In the current study, the efficacy of dietary garlic and onion in regressing preestablished CGS was investigated in experimental mice. After inducing CGS in mice with a lithogenic diet for 10 weeks, they were maintained on basal diets containing 0.6% dehydrated garlic or 2% dehydrated onion for a further 10 weeks. Dietary garlic and onion, either raw or heat processed, regressed preformed CGS in mice up to 53% to 59%, whereas the regression in the basal control diet group was only 10%. The antilithogenic potency of garlic was decreased by its heat processing, but not in the case of onion. Biliary cholesterol was significantly decreased in garlic- and onion-fed animals. Biliary cholesterol saturation index and hydrophobicity index were significantly lowered by dietary garlic and onion. Serum and liver cholesterol levels were decreased by feeding these spices during post-CGS induction period. Hepatic hydroxymethylglutaryl–coenzyme A reductase activity was increased after feeding garlic and onion, whereas activities of the cholesterol-degrading enzymes cholesterol-7 α-hydroxylase and sterol-27-hydroxylase were increased in spice-fed groups. These results indicate that feeding garlic and onion effectively accelerates the regression of preformed CGS by promoting cholesterol desaturation in bile. This observation is significant in the context of evolving dietary intervention strategy to address regression of existing CGS and stopping the possible recurrence.

Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.

The Effects of Sphingolipid Synthesis Inhibition on Cholesterol Gallstone Formation in C57BL/6J mice

Background: Sphingolipids play a very important role in cell membrane formation, signal transduction and plasma lipoprotein metabolism. The first rate-limiting step in the sphingolipid biosynthetic pathway is catalyzed by serine palmitoyltransferase (SPT), and myriocin is a potent and specific inhibitor of SPT. We investigated the impact of SPT inhibition on cholesterol gallstone formation in C57BL/6J mice. Methods: Three groups of eight-week-old C57BL/6J mice were utilized. Each group consisted of 20 mice; group A, B, and C were fed normal chow, lithogenic diet with phosphate buffered saline, and lithogenic diet with myriocin (0.3 mg/kg), respectively, for 6 weeks. The ceramide levels in both serum and bile were assessed by high performance liquid chromatography analysis. Protein expression of ERK, JNK and p38 in the extracted gallbladder were determined by Western-blot analysis. Results: Myriocin treatment caused a significant decrease in the rate of cholesterol gallstone formation. The lithogenic diet mice (group B) showed the highest ceramide activities in both the serum and bile among all the tested groups and there was significant suppression of the ceramide levels in both the serum and bile of the myriocin-treated mice (group C, p < 0.05). Phosphorylation of p38 in the gallbladder was increased in the lithogenic-diet mice and the expression of phosphorylated p38 was significantly suppressed in the myriocin treated mice. Conclusions: SPT inhibition by myriocin suppressed gallstone formation and the levels of ceramide in both the serum and bile. p38 in the cellular signaling pathways might be associated with cholesterol gallstone formation.

Fenugreek seeds reduce atherogenic diet-induced cholesterol gallstone formation in experimental mice

Dietary hypocholesterolemic adjuncts may have a beneficial role in the prevention and treatment of cholesterol gallstones (CGS). In this investigation, fenugreek (Trigonella foenum-graecum) seed was evaluated for this potential on the experimental induction of CGS in laboratory mice. CGS was induced by maintaining mice on a lithogenic diet (0.5% cholesterol) for 10 weeks. Fenugreek seed powder was included at 5%, 10%, and 15% of this lithogenic diet. Dietary fenugreek significantly lowered the incidence of CGS in these mice; the incidence was 63%, 40%, and 10% in the 5%, 10%, and 15% fenugreek groups, respectively, compared with 100% in the lithogenic control. The antilithogenic influence of fenugreek is attributable to its hypocholesterolemic effect. Serum cholesterol level was decreased by 26%-31% by dietary fenugreek, while hepatic cholesterol was lowered by 47%-64% in these high cholesterol-fed animals. Biliary cholesterol was 8.73-11.2 mmol/L as a result of dietary fenugreek, compared with 33.6 mmol/L in high-cholesterol feeding without fenugreek. Cholesterol saturation index in bile was reduced to 0.77-0.99 in fenugreek treatments compared with 2.57 in the high-cholesterol group. Thus, fenugreek seed offers health-beneficial antilithogenic potential by virtue of its favourable influence on cholesterol metabolism.

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.

Effect of vitamin A supplemented diet on calcium oxalate renal stone formation in rats

Objectives To study the effect of a vitamin A supplemented diet on calcium-oxalate stone formation in rats and to test its expected action in the dissolution of renal calculi. Material and methods Twenty-four male Wistar rats were randomly divided into three groups of eight rats each. The first group (group A) received a normal diet for six weeks. The second group (group B) was fed a lithogenic diet by the addition of ethylene glycol 0.5% to drinking water for three weeks then a normal diet for three weeks. The third group (group C) received the same lithogenic diet for three weeks then a vitamin A supplemented diet 20 times the normal amount (5.1 mg/100 g of diet) at the three last weeks. One day before the end of treatment, each animal was placed for 24 h in metabolic cage in order to collect urine samples and determine the urinary parameters. Results The glomerular filtration rate and the urinary excretion of citric acid which fell in group B have been restored in group C. Conclusions This study shows that a vitamin A supplemented diet at the rate of 20 times standard ration could improve the renal function by restoring the glomerular filtration rate and by increasing the urinary pH and excretion of citric acid.

Decreased Expression of Cholesterol 7α-Hydroxylase and Altered Bile Acid Metabolism in Apobec-1−/− Mice Lead to Increased Gallstone Susceptibility

Quantitative trait mapping in mice identified a susceptibility locus for gallstones (Lith6) spanning the Apobec-1 locus, the structural gene encoding the RNA-specific cytidine deaminase responsible for production of apolipoprotein B48 in mammalian small intestine and rodent liver. This observation prompted us to compare dietary gallstone susceptibility in Apobec-1(-/-) mice and congenic C57BL/6 wild type controls. When fed a lithogenic diet (LD) for 2 weeks, 90% Apobec-1(-/-) mice developed solid gallstones in comparison with 16% wild type controls. LD-fed Apobec-1(-/-) mice demonstrated increased biliary cholesterol secretion as well as increased cholesterol saturation and bile acid hydrophobicity indices. These changes occurred despite a relative decrease in cholesterol absorption in LD-fed Apobec-1(-/-) mice. Among the possible mechanisms to account for this phenotype, expression of Cyp7a1 mRNA and protein were significantly decreased in chow-fed Apobec-1(-/-) mice, decreasing further in LD-fed animals. Cyp7a1 transcription in hepatocyte nuclei, however, was unchanged in Apobec-1(-/-) mice, excluding transcriptional repression as a potential mechanism for decreased Cyp7a1 expression. We demonstrated that APOBEC-1 binds to AU-rich regions of the 3'-untranslated region of the Cyp7a1 transcript, containing the UUUN(A/U)U consensus motif, using both UV cross-linking to recombinant APOBEC-1 and in vivo RNA co-immunoprecipitation. In vivo Apobec-1-dependent modulation of Cyp7a1 expression was further confirmed following adenovirus-Apobec-1 administration to chow-fed Apobec-1(-/-) mice, which rescued Cyp7a1 gene expression. Taken together, the findings suggest that the AU-rich RNA binding-protein Apobec-1 mediates post-transcriptional regulation of murine Cyp7a1 expression and influences susceptibility to diet-induced gallstone formation.

Increased susceptibility to diet-induced gallstones in liver fatty acid binding protein knockout mice

Quantitative trait mapping identified a locus colocalizing with L-Fabp, encoding liver fatty acid binding protein, as a positional candidate for murine gallstone susceptibility. When fed a lithogenic diet (LD) for 2 weeks, L-Fabp(-/-) mice became hypercholesterolemic with increased hepatic VLDL cholesterol secretion. Seventy-five percent of L-Fabp(-/-) mice developed solid gallstones compared with 6% of wild-type mice with an increased gallstone score (3.29 versus 0.62, respectively; P < 0.01). Hepatic free cholesterol content, biliary cholesterol secretion, and the cholesterol saturation index of hepatic bile were increased in LD-fed L-Fabp(-/-) mice. Chow-fed L-Fabp(-/-) mice demonstrated increased fecal bile acid (BA) excretion accompanied by decreased ileal Asbt expression. By contrast, there was an increased BA pool and decreased fecal BA excretion in LD-fed L-Fabp(-/-) mice, associated with increased proximal intestinal Asbt mRNA expression, suggesting that intestinal BA absorption was enhanced in LD-fed L-Fabp(-/-) mice. The increase in biliary BA secretion and enterohepatic pool size in LD-fed L-Fabp(-/-) mice was accompanied by downregulation of Cyp7a1 mRNA and increased intestinal mRNA abundance of Fgf-15, Fxr, and Fabp6. These findings suggest that changes in hepatic cholesterol metabolism and biliary lipid secretion as well as changes in enterohepatic BA metabolism increase gallstone susceptibility in LD fed L-Fabp(-/-) mice.

S1777 Toll-Like Receptor 2 ( TLR2) Deficiency Markedly Diminishes Gallstone Formation in C57BL/6 Mice Fed a Lithogenic Diet

S1777 Toll-Like Receptor 2 ( TLR2) Deficiency Markedly Diminishes Gallstone Formation in C57BL/6 Mice Fed a Lithogenic Diet

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders #

TGR5 (Gpbar-1) is a plasma membrane-bound, G protein-coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11-positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate-regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium-dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)-fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist-dependent increase in MQAE-fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. The data suggest a role for TGR5 in bile acid-induced fluid secretion in biliary epithelial cells.

Involvement of osteopontin as a core protein in cholesterol gallstone formation

Matrix proteins are considered to be essential for biomineralization and to be important factors in the formation and growth of gallstones. Osteopontin (Opn) is a noncollagenous, acidic bone-matrix glycoprotein, which is sialated and phosphorylated and which has a cell-binding peptide sequence of glycine-arginine-glycine-aspartate-serine (GRGDS). To investigate the role of Opn in cholesterol gallstone formation, we have studied the involvement of Opn in cholesterol gallstone formation in the human gallbladder wall, in the stones, and in the mouse gallbladder using a gallstone experimental model. Immunohistochemical staining was used in the human gallbladder wall and human gallstones and the determination of mRNA expression by reverse transcriptase-PCR was used in the mouse gallbladder of a gallstone experimental model. The epithelium of stone-laden gallbladders demonstrated high Opn reactivity, as did the core of the stones. Microscopically detected early stones without macroscopic evidence of lithiasis showed the same immunoreactivity as larger stones. Stone-laden gallbladders were infiltrated by macrophages showing intense Opn expression. In gallstone-forming mice, the expression of Opn mRNA and its protein were significantly increased in the gallbladder wall in the early phase of a lithogenic diet intake, before the initiation of inflammation. These results suggest that Opn is possibly involved as a core protein in the formation of cholesterol gallstones.

Dietary garlic and onion reduce the incidence of atherogenic diet-induced cholesterol gallstones in experimental mice

Mice fed with diet containing 0.5 % cholesterol for 10 weeks resulted in cholesterol supersaturation in gallbladder bile which promoted the formation of cholesterol gallstones (CGS). In this study, dietary hypocholesterolaemic spices, garlic and onion (both raw or heat-processed) were examined for their antilithogenic potential by including at 0.6 and 2.0 % level, respectively, along with lithogenic (LG) diet for 10 weeks. Dietary garlic and onion reduced the CGS incidence by 15-39 %, the effect being maximum in the heat-processed onion group. Dietary garlic and onion markedly reduced biliary cholesterol. The cholesterol:phospholipid ratio which was 1.58 in the LG diet group was reduced to 0.73-0.96 in the garlic and onion groups. The biliary cholesterol saturation index was 0.92, 1.25, 1.09 and 0.86, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was 1.9 in the LG group. The hydrophobicity index of bile was - 0.08, - 0.079, - 0.032 and - 0.073, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was +0.054 in the LG group. Hepatic hydroxymethyl glutaryl-CoA reductase activity was lowered in the LG diet-fed group, while dietary garlic or onion countered this alteration and also increased the activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase. Serum and liver cholesterol were decreased by feeding garlic or onion compared to the LG diet. Thus, dietary Allium spices exerted antilithogenic influence by decreasing the cholesterol hyper-secretion into bile and increasing the bile acid output thus decreasing the formation of lithogenic bile in experimental mice.

Shengqing Capsule down-regulates estrogen and progesterone receptors in epithelial tissue of gallbladder in guinea pigs with gallstone

To study the role of estrogen receptor (ER) and progesterone receptor (PR) in the formation of cholesterol calculus and investigate the effects of Shengqing Capsule (SQC), a Chinese patent herbal medicine with the function of soothing liver and draining gallbladder, on ER and PR expressions. A total of 80 female guinea pigs were divided into normal control group, untreated group, ursodeoxycholic acid group (UDCA group) and SQC group. The cholesterol gallstone was induced by feeding the guinea pigs with high-fat lithogenic diet. SQC and UDCA were separately administered to the guinea pigs in the SQC group and UDCA group. After 7-week administration, all the animals were sacrificed to calculate the incidence of calculus formation and detect the expressions the ER and PR in the epithelial tissue of gallbladder by immunohistochemical method. Gallstone was cholesterol calculus detected by infrared spectrum. The incidence of calculus formation in the SQC group (27.78%) was significantly lower than that in the untreated group (81.25%) (X(2)=9.721 5, P=0.001 8). On the basis of Reiner standard, the expression distribution of ER and PR increased gradually from the normal control group through the SQC group and UDCA group to the untreated group. Except for the former two groups and the latter two groups, the differences between the other groups and UDCA group were statistically significant (P<0.05). Besides, the differences of positive expression rates between groups were statistically significant (P<0.05). Increased expressions of ER and PR are closely related to the formation of cholesterol stone. And Shengqing Capsule can down-regulate the expressions of ER and PR.

Cholangiocyte bile salt transporters in cholesterol gallstone–susceptible and resistant inbred mouse strains

We investigated the dietary and gender influences on the expression and functionality of cholangiocyte bile salt transporters and development of biliary hyperplasia in cholesterol gallstone-susceptible C57L/J and resistant AKR/J mice. C57L and AKR mice were fed chow, a lithogenic diet, or a cholic acid-containing diet for 14 days. Expression of cholangiocyte bile salt transporter proteins ASBT (SLC10A2), ILBP (FABP6), and MRP3 (ABCC3) were studied by Western blot analysis. Taurocholate uptake studies were performed using microperfusion of isolated bile duct units. The pre- and post-perfusion taurocholate concentrations were analyzed by high performance liquid chromatography. Biliary proliferation in liver sections was scored. The lithogenic diet induced ductular proliferation in C57L mice. On chow, SLC10A2 and ABCC3 were overexpressed in male and female C57L compared to AKR mice. A lithogenic diet reduced the expressions of FABP6 in both male and female C57L mice, SLC10A2 in female C57L mice, and ABCC3 in male C57L mice. These alterations in transporter expressions were not associated with changes in taurocholate uptake. The lithogenic diet induced biliary hyperplasia and reduced bile salt transporter expressions in C57L mice. Although bile salt uptake was not increased in the bile duct unit, we speculate that the biliary hyperplasia on the lithogenic diet may lead to an increase in intrahepatic bile salt recycling during cholesterol cholelithogenesis.

Ezetimibe prevents cholesterol gallstone formation in mice

Intestinal cholesterol absorption may influence gallstone formation and its modulation could be a useful therapeutic strategy for gallstone disease (GSD). Ezetimibe (EZET) is a cholesterol-lowering agent that specifically inhibits intestinal cholesterol absorption. To test whether EZET can prevent gallstone formation in mice. Gallstone-susceptible C57BL/6 inbred mice were fed control and lithogenic diets with or without simultaneous EZET administration. Lithogenic diet increased biliary cholesterol content and secretion, and induced sludge or gallstone formation in 100% of the animals. EZET administration reduced intestinal cholesterol absorption by 90% in control animals and by 35% in mice receiving the lithogenic diet. EZET prevented the appearance of cholesterol crystals and gallstones. In addition, mice fed the lithogenic diet plus EZET exhibited a 60% reduction in biliary cholesterol saturation index. Of note, EZET treatment caused a significant increase in bile flow (+50%, P<0.01) as well as bile salt, phospholipid and glutathione secretion rates (+60%, +44% and +100%, respectively, P<0.01), which was associated with a moderately increased expression of hepatic bile salt transporters. In addition, relative expression levels of Nieman-Pick C1 like 1 (NPC1L1) in the enterohepatic axis in humans were assessed. Expression levels of NPC1L1 were 15- to 30-fold higher in the duodenum compared with the liver at transcript and protein levels, respectively, suggesting preferential action of EZET on intestinal cholesterol absorption in humans. In a murine model of GSD, EZET prevented gallstone formation by reducing intestinal cholesterol absorption and increasing bile salt-dependent and -independent bile flow. EZET could be useful in preventing GSD disease in susceptible patients.

Hepatic insulin resistance directly promotes formation of cholesterol gallstones

Despite the well-documented association between gallstones and the metabolic syndrome, the mechanistic links between these two disorders remain unknown. Here we show that mice solely with hepatic insulin resistance, created by liver-specific disruption of the insulin receptor (LIRKO mice) are markedly predisposed toward cholesterol gallstone formation due to at least two distinct mechanisms. Disinhibition of the forkhead transcription factor FoxO1, increases expression of the biliary cholesterol transporters Abcg5 and Abcg8, resulting in an increase in biliary cholesterol secretion. Hepatic insulin resistance also decreases expression of the bile acid synthetic enzymes, particularly Cyp7b1, and produces partial resistance to the farnesoid X receptor, leading to a lithogenic bile salt profile. As a result, after twelve weeks on a lithogenic diet, all of the LIRKO mice develop gallstones. Thus, hepatic insulin resistance provides a crucial link between the metabolic syndrome and increased cholesterol gallstone susceptibility.